Lubman Olga Y, Ilagan Ma Xenia G, Kopan Raphael, Barrick Doug
T.C Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA.
J Mol Biol. 2007 Jan 19;365(3):577-89. doi: 10.1016/j.jmb.2006.09.071. Epub 2006 Oct 3.
Complex formation between the intracellular domain of the Notch receptor (NICD) and the transcription factor CSL is indispensable for transcriptional activation. To understand how NICD displaces CSL-associated co-repressors, we have quantified the binding of different Notch1 ICD regions to a key interaction domain (the beta trefoil domain, or BTD) of human CSL. Electrophoresis, scattering, and titration calorimetry indicate that NICD and BTD combine to form a 1:1 heterodimer. Neither the Notch1 ankyrin domain (ANK) nor C-terminal region contributes binding energy towards BTD. In contrast, binding energy is attributed largely to a short segment including the conserved WFP sequence motif within the RAM region (the approximately 140 residue polypeptide segment N-terminal to the ANK domain); substitution of this motif substantially reduces affinity. Short (< or =25 residues) WFP-containing peptides encoded by the four mammalian Notch genes have similar affinities to BTD; thus, activity differences between paralogues either result from other regions of NICD and CSL or from differences in interaction with downstream components. The importance of RAM was demonstrated by the ability of a short RAM peptides to dissociate NICD:CSL interaction in cellular lysates. These results support an emerging molecular mechanism for the displacement of co-repressors from DNA-bound CSL by NICD.
Notch受体的细胞内结构域（NICD）与转录因子CSL之间形成复合物对于转录激活是必不可少的。为了了解NICD如何取代与CSL相关的共抑制因子，我们已经对不同的Notch1 ICD区域与人类CSL的关键相互作用结构域（β三叶形结构域，或BTD）的结合进行了定量分析。电泳、散射和滴定热分析表明，NICD和BTD结合形成1:1异二聚体。Notch1锚蛋白结构域（ANK）和C末端区域都不会对BTD贡献结合能。相反，结合能主要归因于一个短片段，该片段包括RAM区域内保守的WFP序列基序（ANK结构域N端约140个残基的多肽片段）；该基序的取代会大大降低亲和力。由四个哺乳动物Notch基因编码的短（≤25个残基）含WFP肽与BTD具有相似的亲和力；因此，旁系同源物之间的活性差异要么源于NICD和CSL的其他区域，要么源于与下游成分相互作用的差异。短RAM肽能够在细胞裂解物中解离NICD:CSL相互作用，这证明了RAM的重要性。这些结果支持了一种新出现的分子机制，即NICD将共抑制因子从与DNA结合的CSL上置换下来。
